Open Access Full Text Article Evaluation of Human

OR I G I N A L R E S E A R C H

Evaluation of Human Esophageal Epithelium

Permeability in Presence of Different Formulations

Containing Hyaluronic Acid and Chondroitin

SulphateThis article was published in the following Dove Press journal:

Medical Devices: Evidence and Research

Gaia Pellegatta1

Marco Spadaccini1

Laura Lamonaca1

Vincenzo Craviotto1

Ferdinando D’Amico1

Laura Ceriotti2

Marisa Meloni 2

Alessandro Repici 1

1Humanitas Clinical and Research Center

and Humanitas University, Digestive

Endoscopy Unit, Division of

Gastroenterology, Rozzano, MI, Italy;2VitroScreen, Milano, Italy

Purpose: New medical devices that contain hyaluronic acid (HA) and chondroitin sulphate

(CS), with or without antacid components, have been developed for the treatment of

gastroesophageal reflux disease (GERD) with the aim of improving oesophageal mucosal

defences by creating a film on the oesophageal mucosa and acting as a mechanical barrier

against the noxious components of refluxate, both acidic and basic.

Methods: The film-forming and protective efficacy of medical device A based on HA

and CS plus aluminium hydroxide, device B combining HA and CS with magnesium

trisilicate and device C with only the combination of HA and CS was tested on

a reconstructed human oesophageal epithelium (HO2E/S/5) as a biological model in 2

different pH environments, neutral and acidic, to mimic realistic conditions. Caffeine

penetration kinetics and Lucifer yellow (LY) permeability modifications induced by these

products were compared to those induced by a negative control series (saline solution,

code NC) and positive control series (white Vaseline, code V) under neutral and acidic

pH conditions.

Results: Under neutral and acidic pH conditions, compared to the negative control, all the

products tested reduced (>80% and 85–90%, respectively) the caffeine passage, and no

significant difference was observed among the products tested. Under neutral and acidic

conditions, the LY permeabilities registered with device A and device C were not different

from that registered with the negative control, while an LY flux% increase was calculated

after 2 hrs of treatment (21.1%) with device B under acidic conditions.

Conclusion: These results confirm the ability of the products tested to interact with the

oesophageal epithelium in order to adhere and create a stable protective film for at least 2

hours after their homogeneous distribution on the epithelium surface. Further clinical

studies are needed to test these devices in the topical treatment of gastroesophageal reflux

symptoms.

Keywords: hyaluronic acid, chondroitin sulphate, gastroesophageal reflux disease, antacid,

caffeine, Lucifer yellow

IntroductionIn recent years, the development of drugs capable of acting on pathophysiological

mechanisms other than acid has been evaluated for the treatment of gastroesopha-

geal reflux disease (GERD), non-erosive reflux disease (NERD) and proton pump

inhibitor (PPI)-non-responder patients. Great attention has been paid to the use of

Correspondence: Gaia PellegattaDigestive Endoscopy Unit, Division ofGastroenterology, Humanitas ResearchHospital, Via Manzoni 56, Rozzano, MI20089, ItalyTel +390282247091Email [email protected]

Medical Devices: Evidence and Research Dovepressopen access to scientific and medical research

Open Access Full Text Article

submit your manuscript | www.dovepress.com Medical Devices: Evidence and Research 2020:13 57–66 57

http://doi.org/10.2147/MDER.S234810

DovePress © 2020 Pellegatta et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing

the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).

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those agents that aim to potentiate the defensive properties

of oesophageal mucosa with or without the association of

antacid components.

The resistance of oesophageal mucosa is due to multi-

ple factors, which can be grouped into three categories: 1)

pre-epithelial (salivary secretion, secretion of muco-

bicarbonates); 2) epithelial (stratified cells of squamous

epithelium that reduce the retro-diffusion of hydrogen

ions and favour their neutralization); and 3) post-

epithelial (mainly the mucosal blood supply responsible

for the (mechanisms of) cellular repair).

In the last few years, new medical devices that contain

hyaluronic acid (HA) and chondroitin sulphate (CS) with

or without antacid components have been developed to

ameliorate oesophageal mucosal defences by creating

a film on the oesophageal mucosa and acting as

a mechanical barrier against the noxious components of

refluxate, both acidic and basic.

HA and CS are two important components of many

extracellular matrices and are involved in several important

physiological processes, such as wound repair and regen-

eration, cell proliferation, tissue morphogenesis and matrix

organization. HA is a high-molecular-weight glycosamino-

glycan and a component of the majority of extracellular

matrices that play a key role in tissue healing. In this

process, HA is crucial for a range of different activities,

including the activation and moderation of the inflammatory

response; the promotion of cell proliferation, migration and

angiogenesis; the promotion of re-epithelization via the

proliferation of basal keratinocytes; and the reduction in

collagen deposition that leads to scarring.1 Clinical studies

have shown that topical application of HA helps the healing

process of venous leg ulcers,2 reduces the incidence of

high-grade radio-epithelitis in patients who have undergone

radiotherapy for head and neck, breast or pelvic

carcinomas,3 and brings fast symptom relief in recurrent

aphthous ulcerations of the oral mucosa.4

CS is a natural glycosaminoglycan that is present in the

extracellular matrix of skin, cartilage, ligaments and ten-

dons. In the literature, there are data available regarding

the anti–inflammatory, tissue morphogenesis, cell prolif-

eration and wound repair properties of CS.5 These effects

are related to the capacity of CS to interact with a wide

variety of molecules, including matrix molecules, growth

factors, protease inhibitors, cytokines, chemokines, and

adhesion molecules.6 As both in vitro and in vivo experi-

mental studies have shown that CS specific binding to

pepsin reduces peptic activity,7,8 treatments of peptic

ulcer with CS have been attempted in the past.9,10 The

negative effect of pepsin on oesophageal mucosa may also

be neutralized by CS.

The aim of this study was to test the film-forming

properties and protective properties of different medical

devices, solid tablets containing HA and CS, on a 3D

reconstructed human oesophageal epithelium (HO2E/S/5)

as a biological model under neutral (pH=7) and acidic

(pH=3.3) pH conditions.

In vitro reconstructed human epithelial models are

similar in terms of morphology (multi-stratified or epithe-

lium) and biochemical and physiological properties to

in vivo human tissues, and currently, they represent the

most promising alternative to animal ex vivo explants and

submerged cell monolayers as biological models to be

adopted in preclinical research.11

The availability of organ-specific biological barrier

models is an advantage in preclinical testing: the status,

functionality and reproducibility of these barriers are key

parameters for assessing the biological effects of medical

devices on the part of the body where they are intended to

exert their action and allow discrimination between differ-

ent formulations. Furthermore, the use of reconstructed

human 3D tissues presents a series of advantages over

traditional tests performed on cell monolayers or in vivo:

1. overcome the limited availability of ex vivo explants

(ex vivo mucosae are not even available) and ethical

concerns regarding ex vivo explants of human and

animal origin: the 3D models are commercially avail-

able, are produced under standardized conditions and

are quality controlled;

2. provide a standardized, robust and reproducible

model of epithelia and mucosae of human origin

with well-characterized and quantifiable barrier

properties (eg, cell viability, barrier permeability,

biomarkers);

3. provide data that are more predictive of human

responses through the use of experimental protocols

using tissue models, which can reduce toxicological

risks. An essential advantage attributable to in vitro

3D human models is the possibility of evaluating

products directly on an organized tissue with differ-

ent cell layers at the same doses used in vivo after

acute and repeated exposures.

Last but not least, these models are sustainable with respect to

the European Directive n. 2010/63, which promotes the

Pellegatta et al Dovepress

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DovePressMedical Devices: Evidence and Research 2020:1358

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development and validation of alternative methods under the

Principle of Replacement, Reduction, and Refinement (3Rs)

of the use of animals in research and foresees the replacement

of animal testing for scientific and educational purposes once

alternatives are scientifically available. This Directive has had

a transversal impact on many European legislations dealing

with the safety and market entry of chemicals and consumer

products (eg, cosmetics, medicines, biocides, etc.) and more

recently has been explicitly endorsed by the EuropeanMedical

Device Regulation n. 2017/745 (MDR).

We mean “film-forming” as protective properties, there-

fore a modification of epithelial permeability where the

product acts as a physical barrier to a probe transport across

the tissue, resulting in a protective physical barrier (“film”)

on the epithelium. The film-forming and protective efficacy

of a medical device based on HA and CS plus aluminium

hydroxide (univocal code A, HA:AH:CS are in the ratio

1:20:40), a device combining HA and CS with magnesium

trisilicate (univocal code B HA:MT:CS are in the ratio

1:20:40) and a device with only the combination of HA

and CS (univocal code C HA:CS are in the ratio 1:40) was

tested on a reconstructed human oesophageal epithelium

(HO2E/S/5) as a biological model at different pH (neutral

and acidic conditions), thereby mirroring different degrees

of tissue damage and epithelial permeability.

To mimic realistic exposure conditions and the pro-

ducts’ interaction with physiological fluids (i e saliva), an

optimized procedure was adopted: according to the pro-

duct’s technical forms (powder), the products were dosed

as powder obtained from the solid tablets in a previously

wetted epithelium (saline solution) in an amount adapted

to cover the whole epithelium surface with a homogeneous

distribution for 2 hours at room temperature. Caffeine

penetration kinetics and Lucifer yellow (LY) permeability

modifications induced by these products were compared to

those induced by a negative control series (saline solution,

code NC) and positive control series (white Vaseline, code

V) under neutral and acidic pH conditions. Figure 1 shows

the in vitro study protocol.

Materials and MethodsTissue ModelThe human oesophageal epithelium (HO2E/S/5) is an

epithelium formed after 5 days of airlift culture of the

K510 cell line (the final product Oesophageal Epithelium

was purchased from Episkin) on inert polycarbonate filters

in a chemically defined medium that reproduces the human

oesophageal epithelium morphology. The reconstructed

human oesophageal epithelium used for this study had

a size of 0.5 cm2 and a thickness of at least 80 µm. The

morphological structure and its similarity with the human

oesophageal tissue are described in Figure 2.

The batch was tested for the absence of hepatitis B,

hepatitis C and mycoplasma, and the maintenance medium

was tested for sterility.

The inserts containing the tissues at day 5 were placed

at room temperature in a multi-well plate filled with an

agarose nutrient solution in which they were embedded.

The HO2E/S/5 was then removed from the agarose

nutrient solution under a sterile airflow cabin. The inserts

were rapidly transferred to 4-well plates previously filled

with maintenance medium (1 mL/well) at room temperature

and incubated at 37°C, 5% CO2 and saturated humidity.

The day after, the medium was changed, and after the

wetting-moisturizing procedure necessary to better recapi-

tulate the realistic interaction of the medical device dis-

persed in the oesophageal environment, 30 μL of controls

(saline solution NaCl 90%, Vaseline) and 30 mg of the test

items (GERDOFF, B, C) were directly and uniformly

applied topically on the 3D model epithelium to mimic

realistic exposure conditions and the interaction with phy-

siological fluids (i e, saliva).

Test ItemsAll the test items are solid compounds formulated in melt-

in-mouth tablets (Table 1).

After 15 minutes of treatment, 100 μL of 0.5% caffeine

solution in bi-distilled water (acid or standard) was applied

for 2 hours on pre-wetted epithelium without washing out

Figure 1 Scheme of the in vitro experimental design.

Dovepress Pellegatta et al

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the product. To evaluate the kinetics of caffeine permeabil-

ity, receptor fluid in the basolateral compartment (1 mL of

saline solution) was collected after 15 mins, 1 hour and

2 hours, and the caffeine content was quantified by the

UPLC method. After 2 hours of caffeine application and

after receptor fluid collection, the pH of the apical compart-

ment was measured (with litmus precision paper range

4.9–9.5), and then the Lucifer yellow assay was performed

immediately.

Caffeine Permeability EvaluationFor studies of percutaneous absorption, the OECD guide-

line 428 recommends the use of caffeine (MW = 194.2,

logP o/w = - 0.08) as the reference compound at low

lipophilicity. This molecule has been used in several per-

cutaneous absorption studies to evaluate permeability

using different models of epidermis reconstructed in vitro.

Due to the ability to overcome the epithelial barrier,

even in the absence of damage, caffeine was used as

a probe to assess the propensity of a given product to

form a protective film: the reduction in the passage of

caffeine through the biological model is used as an index

of film-forming efficacy. Given its film-forming and occlu-

sive properties, Vaseline was used as a positive control.

Caffeine Penetration ProtocolThe culture inserts were placed in 6-well plates previously

filled with 1 mL/well of saline solution (basolateral

compartment, receptor fluid). Controls and test items were

evaluated in triplicate. Thirty milligrams of test items and

30 μL of saline solution (negative control) and white

Vaseline (positive control) were applied on the apical sur-

face of pre-wetted HO2E/S/5 tissues for 15 minutes; this

time was defined after a preliminary assay that confirmed

a homogeneous distribution on the epithelial surface. Then,

0.1 mL (100 μL) of caffeine solution (0.5% w/v (1 mg

caffeine/cm2)) under neutral (pH 7) or acidic (pH 3.3)

conditions was applied to the treated epithelial surface.

Throughout the experiment, the inserts were placed in

a CO2 incubator at a defined temperature. After 15 minutes,

1 and 2 hours, 1 mL of the receptor fluid was withdrawn

from the basolateral compartment with a micropipette

(Gilson) and replaced with fresh receptor fluid kept at

room temperature. The withdrawn samples were stored at

4°C before UPLC/MS analysis. Figure 3 shows the caf-

feine penetration protocol.

At the end of the experiment, the pH of the solution in

the apical compartment was measured (with litmus preci-

sion paper range 4.9–9.5), and then the Lucifer yellow

assay was performed immediately.

Analytical Method for CaffeineThe caffeine concentration was determined by using

a 1290 infinity II LC System (AGILENT Santa Clara

California) equipped with a C18 reversed-phase column

(ACQUITY UPLC BEH-C18, 1.7 μm, 100 x 2.1 mm,

WATERS CORPORATION, Massachusetts) set at

25°C. A 5 μL sample was injected for isocratic elution

at 0.25 mL/min. The composition of the eluent was 80%

water/20% methanol. The wavelength was set at

273 nm. Standard calibration curves for caffeine (0.1

and 1000 mg/L) were used.

Lucifer Yellow AssayLucifer yellow (LY) is a fluorescent dye impermeable to

the cell membrane and is used to study the paracellular

A B

Figure 2 Morphological structure of human 3D reconstructed oesophageal epithelium (A) and its similarity with the biopsy of human full-thickness oesophageal mucosa (B).

Table 1 Test Item Characterization

Negative

Control

Saline Solution NaCl 90%

Positive control Vaseline

Test items Device A: GERDOFF, based on HA, CS, aluminium

hydroxide

Device B: HA, CS, magnesium trisilicate

Device C: HA, CS




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permeability of a substance. When the junctions are

unbroken, LY has a very low permeability; if the joints

are damaged, LY flow will be much higher. Therefore,

this assay is used to verify the integrity of cell junctions

in the presence of the substance that needs to be

evaluated.

Lucifer Yellow ProtocolThe LY flux was evaluated after the caffeine permeabil-

ity assay and the removal of test items from the surface

of the tissues. First, 0.5 mL of LY (500 μM in saline

solution) was applied to the epithelial surface, and 1 mL

of saline solution was added to the basolateral compart-

ment. After 1 hr at 37°C, the relative passage of LY

from the apical to the basolateral compartment was

quantified.

The measurement of fluorescence (RFU) was per-

formed in a spectrofluorimeter (TECAN INFINITE

M200) with 428 nm excitation and 535 nm emission. For

each tissue, the measurement was performed at the baso-

lateral level, and flux was calculated with the following

formula:

LY Flux % ¼ RFUBL=RFUAP t¼0ð Þ x 100

where BL=basolateral and AP t=0: mean of the RFU of

LY 500 μM solution.

ResultsCaffeine Quantification Under Neutral

pH ConditionsThe results of caffeine passage under neutral conditions

(pH 7) are reported in Tables 2–4. Table 2 reports micro-

grams of caffeine quantified in the receptor fluids under

neutral conditions expressed as the mean of biological

triplicate assays ± standard deviation and coefficient of

variation (CV%).

Figure 3 Caffeine penetration protocol.

Table 2 Micrograms of Caffeine Quantified in the Receptor Fluid

at 15 mins, 1 hr and 2 hrs Under Neutral Conditions

Caffeine µg in Basolateral Compartment

15 mins

(CV%)

1 hr

(CV%)

2 hrs

(CV%)

Negative

Control

89.84 ± 2.88

(3.2)

152.94 ± 0.65

(0.4)

123.66 ± 9.04

(7.3)

Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.28 ± 0.00

(1.3)

17.66 ± 0.17

(1.0)

35.07 ± 0.13

(0.4)

Device B 0.89 ± 0.02

(2.5)

26.59 ± 0.09

(0.3)

42.87 ± 0.32

(0.7)

Device C 0.29 ± 0.00

(0.8)

17.26 ± 0.14

(0.8)

37.55±0.05

(0.1)

Note: *Value below 0.1 μg.



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The total amount quantified in the negative control

(366.44 μg ± 9.66 μg) was considered as a reference for

caffeine kinetics on HO2E/S/5 under the adopted neutral

experimental conditions. As expected, Vaseline completely

inhibited caffeine passage (0.00 ± 0.00). These results

allow us to validate the experiment.

In Table 3, the results under neutral conditions are

expressed as the percentage of caffeine considering the

dose applied (0.5 mg) as 100%.

In Table 4 and Figure 4, the results are expressed as

the percentage of caffeine considering the dose quanti-

fied when the negative control was applied (366.44 μg)

as 100%.

Compared to the negative control, all the products

tested reduced the caffeine passage by >80%. At each

time point, no significant difference was observed among

the products tested.

Caffeine Quantification in Acidic pH

ConditionsThe results of the caffeine passage under acidic conditions

(pH 3.3) are reported in Tables 5–8.

Table 5 reports the micrograms of caffeine quantified

in the receptor fluid under acidic conditions expressed as

the mean ± standard deviation and coefficient of variation

(CV%). The total amount quantified in the negative con-

trol (365.33 μg ± 2.93 μg) was considered the reference of

caffeine kinetics on HO2E/S/5 under the adopted acid

experimental conditions. It is important to emphasize that

despite the treatment with an acidic caffeine solution, no

significant difference from negative control tissues

exposed to neutral conditions (366.44 μg ± 9.66 μg) was

observed. As expected, Vaseline completely inhibited the

caffeine passage (0.00 ± 0.00), and these results allow us

to validate the experiment.

Table 3 Rate of Caffeine Quantified in the Receptor Fluid at

15 mins, 1 hr and 2 hrs Under Neutral Conditions

Caffeine % in Basolateral Compartment

Compared to the Dose Applied

15 mins 1 hr 2 hrs

Negative Control 18.0 ± 0.6 30.6 ± 0.1 24.7 ± 1.8

Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.1 ± 0.0 3.5 ± 0.0 7.0 ± 0.0

Device B 0.2 ± 0.0 5.3 ± 0.0 8.6 ± 0.1

Device C 0.1 ± 0.0 3.5 ± 0.0 7.5 ± 0.0


Table 4 Rate of Caffeine Considering the Caffeine Quantified

After 2 hrs When the Negative Control Was Applied Under

Neutral Conditions as 100%


Compared to the Negative Control

15 mins 1 hr 2 hrs Total

Negative Control 24.5 ± 0.8 41.7 ± 0.2 33.7 ± 2.5 100.0 ± 2.6

Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.1 ± 0.0 4.8 ± 0.0 9.6 ± 0.0 14.5 ± 0.1

Device B 0.2 ± 0.0 7.3 ± 0.0 11.7 ± 0.1 19.2 ± 0.1

Device C 0.1 ± 0.0 4.7 ± 0.0 10.2 ± 0.0 15.0 ± 0.1


0.0

20.0

40.0

60.0

80.0

100.0

120.0

15 min 1 h 2 h

Caf

fein

e %

Caffeine % under neutral conditions compared to Negative Control

NC Device A Device B Device C

Figure 4 Rate of caffeine considering the caffeine quantified after 2 hrs when the negative control (NC) was applied under neutral condition as 100%.




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In Table 6, the results under acidic conditions are

expressed as the percentage of caffeine considering the

dose applied (0.49 mg) as 100%.

In Table 7 and Figure 5, the results are expressed as the

percentage of caffeine considering the dose quantified

when the negative control was applied (365.33 μg) as

100%. Compared to the negative control, all the products

tested reduced the caffeine passage significantly (85–90%

reduction). At each time point, no significant difference

was observed among the products tested.

Lucifer YellowLY paracellular flux was assessed at the end of the expo-

sure time (2 hrs). The measured fluorescence results are

reported in Figure 6 (pH 7 condition) and Figure 7 (pH 3.3

condition).

Figure 6 shows the LY flux % with respect to the

amount of LY in the apical compartment at time 0. The

negative control (NC) had an LY permeability of 5.6%

after 2 hrs of treatment.

The LY permeabilities registered after 2 hours with

device A and device C were not different from that regis-

tered with the negative control. These results indicate, for

the products tested, an absence of direct damage at the tight

junction level and the maintenance of barrier functionality

after exposure.

In contrast, an LY flux% increase was observed

(12.9%) with device B.

Figure 7 shows the LY flux % with respect to the

amount of LY in the apical compartment at time 0 under

acidic conditions.

Confirming the results observed under neutral condi-

tions, the LY permeabilities registered after 2 hours with

device A and device C were not different from that regis-

tered with the negative control, while a LY flux % increase

(21.1%) was calculated with device B.

pH MeasurementAfter 2 hours of caffeine application and after receptor

fluid collection, the pH of the apical compartment was

measured (with litmus precision paper range 4.9–9.5).

Table 8 reports the results.

Table 5 Micrograms of Caffeine Quantified in the Receptor Fluid

at 15 mins, 1 hr and 2 hrs Under Acidic Conditions

Caffeine µg in Basolateral Compartment

15 mins

(CV%)

1 hr

(CV%)

2 hrs

(CV%)

Negative Control 118.42 ± 0.07

(0.1)

147.58 ± 1.82

(1.2)

99.32 ± 1.37

(1.4)

Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.59 ± 0.10

(17.7)

9.74 ± 0.02

(0.2)

18.97 ± 0.07

(0.4)

Device B 0.66 ± 0.00

(0.7)

14.39 ± 0.05

(0.3)

25.48 ± 0.05

(0.2)

Device C 2.02 ± 0.01

(0.6)

21.30 ± 0.10

(0.5)

33.17 ± 0.01

(0.0)


Table 6 Rate of Caffeine Quantified in the Receptor Fluid at

15 mins, 1 hr and 2 hrs Under Acidic Conditions


Compared to Dose Applied

15 mins 1 hr 2 hrs


Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.1 ± 0.0 2.0 ± 0.0 3.9 ± 0.0

Device B 0.1 ± 0.0 2.9 ± 0.0 5.2 ± 0.0

Device C 0.4 ± 0.0 4.3 ± 0.0 6.8 ± 0.0


Table 7 Rate of Caffeine Considering the Caffeine Quantified

After 2 hrs When the Negative Control Was Applied Under

Acid Conditions as 100%


Compared to Negative Control

15 mins 1 hr 2 hrs


Vaseline 0.0 ± 0.0* 0.0 ± 0.0* 0.0 ± 0.0*

Device A 0.2 ± 0.0 2.7 ± 0.0 5.2 ± 0.0

Device B 0.2 ± 0.0 3.9 ± 0.0 7.0 ± 0.0

Device C 0.6 ± 0.0 5.8 ± 0.0 9.1 ± 0.0


Table 8 pH Measurements

pH Value Measured in the Apical

Compartment

Neutral Condition Acidic Condition

Negative Control 5.0 5.0

Vaseline 5.0 4.9

Device A 6.3–6.6 6.8–6.9

Device B 8.8 8.8

Device C 7.2 7.0



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0.0

20.0

40.0

60.0

80.0

100.0

120.0

15 min 1 h 2 h

Caf

fein

e %

Caffeine % in acidic conditions compared to Negative Control

NC Device A Device B Device C

Figure 5 Rate of caffeine considering the caffeine quantified after 2 hrs in the negative control as 100% under acidic conditions.

NC V Device A Device B Device C2 h 5.6 5.5 5.2 12.9 5.4

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Luc

ifer

yel

low

Flu

x %

Lucifer yellow at 2 h: pH 7 condition

Figure 6 Lucifer yellow flux after 2 hrs of treatment followed by product washing under neutral conditions.

NC V Device A Device B Device C2 h 8.7 9.1 8.8 21.1 8.9

0.0

5.0

10.0

15.0

20.0

25.0

Luc

ifer

yel

low

Flu

x %

Lucifer yellow at 2 h: pH 3.3 condition

Figure 7 Luciferase yellow flux after 2 hrs of treatment followed by product washing under acidic conditions.




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Compared to the negative control, all the tested pro-

ducts induced a pH increase under both conditions. In

particular, with device B, a basic value of 8.8 was reached.

This observation directly correlates and explains the

observed LY flux increase.

DiscussionThe human esophageal epithelium permeability and prop-

erties of device A, device B and device C were tested on

a reconstructed human oesophageal epithelium (HO2E/S/

5) as a biological model under different pH conditions:

neutral (pH 7.0) and acidic (pH 3.3).

The adopted experimental conditions mirror realistic

exposure in the oesophageal cavity environment: the pre-

sence of a significant liquid volume in contact and sur-

rounding the epithelium. To mimic neutral (healthy) and

acidic (disease) conditions, two different pH conditions

were investigated.

Caffeine penetration kinetics and LY permeability

modifications induced in the product-treated series were

compared to those induced in the negative control series

(saline solution) and the positive control series (white

Vaseline) under both the pH conditions tested.

Compared to the negative control, all the products

tested significantly reduced the caffeine passage, and at

each time point, no significant difference was observed

among the different products tested.

These results confirm the ability of these products to

interact with the oesophageal epithelium and to adhere and

create a stable protective film for at least 2 hours after their

homogeneous distribution on the epithelium surface in the

presence of a relatively high amount of liquid, represented by

the caffeine solutions applied to the apical surface (100 μL).The oesophageal mucosa is protected against injurious

agents by its stratified, multi-layered squamous epithelium,

which represents a true mucosal barrier. All the damaging

substances, such as the hydrochloric acid and pepsin con-

tained in the gastric refluxate, may impair this barrier and

as a consequence may increase the mucosal permeability.

The caffeine permeation assay showed that no modifi-

cations of the physiological permeability of the epithelium

were induced by the tested products. This result confirms

that the human esophageal epithelium permeability and

protective properties under the adopted experimental con-

ditions were exclusively related to physiological interac-

tion and protective activity of the epithelium, which

maintained its functional role. The results of the LY

assay show that after exposure to the negative control,

Vaseline, and devices A and C, there was no alteration in

the paracellular permeability of the 3D model. In fact,

devices A and C and Vaseline create a protective barrier

that is positioned above the epithelium without modifying

its structure and permeability. The application of device B,

which contains magnesium trisilicate, an anti-acidic pro-

duct, in addition to HA and CS leads to an increase in the

permeability of the 3D epithelium under both neutral and

acidic pH conditions. These results suggest that this pro-

duct had an impact at the tight junction level and on

epidermal barrier functionality.

To our knowledge, this work is the first study in the

literature on the use of 3D human tissue models applied to

oesophageal mucosa and on the efficacy and mechanism of

action of products CS and HA at different pH (neutral and

acidic conditions) mirroring different degrees of tissue

damage and permeability.

To date, few studies in the literature have shown the

effects of products containing CS and HA on the relief of

GERD symptoms. Two small prospective placebo-controlled

studies have shown that short-term treatment achieved sig-

nificant and rapid symptom relief in patients with both

erosive reflux disease12 and NERD.13 More recently,

a prospective double-blind placebo-controlled trial con-

ducted in several Italian centres showed that the combination

of a PPI + HA-CS in syrup was able to relieve symptoms and

improve the quality of life more than a PPI alone.14

A recent open-label uncontrolled study showed that

administration of orodispersible tablets containing CS,

aluminium hydroxide and HA improves non-erosive

GERD clinical typical and atypical symptoms and gastric

juice-related biochemical parameters (eg, neutrophil, lym-

phocyte, eosinophil, parietal cells, red blood cells and

exudate protein counts).15

Recently, 3D tissue models have been adopted in the field

of medical devices for the evaluation of potential irritation

and sensitization by medical device extracts on the skin.16–18

This work is the first study in which a 3D oesophageal tissue

model, formed from a human cell line culture, is used for

testing medical device mechanisms of action. This experi-

mental model brings a great advantage because of the pos-

sibility of evaluating the effectiveness of different products

on an organized tissue with different cell layers at the same

doses used in vivo under different conditions.

In conclusion, all solid formulations tested showed

good film-forming properties on a reconstructed human

oesophageal epithelium independent of the presence of

an antacid in the formulation, mirroring different degrees



DovePress65

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of epithelial permeability (neutral or acidic pH), and they

showed a positive effect on enhancing the barrier integrity

under both conditions tested.

DisclosureCL and MM are full employees of Vitroscreen. The

authors report no other conflicts of interest in this work.

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https://doi.org/10.1046/j.1524-475X.1999.00079.x

https://doi.org/10.1046/j.1524-475X.1999.00079.x

https://doi.org/10.3109/09546639609089533

https://doi.org/10.1016/S0167-8140(96)01882-8

https://doi.org/10.1111/jop.2006.35.issue-8

https://doi.org/10.2174/157016308786733564

https://doi.org/10.1007/s10787-011-0098-0

https://doi.org/10.1111/apt.13914

https://doi.org/10.1016/j.tiv.2013.08.006





http://www.dovepress.com/testimonials.php

http://www.dovepress.com/testimonials.php



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